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To use the Primer3, open a DNA sequence and select the Analyze ‣ Primer3... context menu item. The dialog will appear:
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However there is one additional feature available which is not originally a part of Primer3 tool. It allows user design primers for RT-PCR experiments by choosing which exons/introns to span with the primer product. This feature is described in detailed below.
RTPCR primer design
This feature allows to span corresponding intron sequences on the genomic sequence or junction sequences between exons.
Overview
Note: ths feature is only available for mRNA/cDNA sequences with annotated exons. There are several ways to obtain the cDNA for a corresponding DNA sequence.
- From NCBI or ENSEMBLE database
- Align the genomic and cDNA sequences using spliced aligner
Options
Exon annotation name
Span exon-exon junction
Exon range
Span at least one intron: the default mode